Application of interspersed repetitive sequence polymerase chain reaction for construction of yeast artificial chromosome contigs.
Construction of physical maps across candidate regions is one of the rate-limiting steps of positional cloning projects. To date, most physical maps have been constructed by polymerase chain reaction (PCR)-based sequence-tagged site (STS) content mapping. While effective, this technique has a number of disadvantages including problems with yeast artificial chromosome (YAC) chimerism, the time and effort required to generate new STSs from YAC ends, the cost of primer synthesis for large contiging projects, and the time, effort, and expense necessary for screening each STS in the two-tiered hierarchical YAC library screening format. An alternative strategy, interspersed repetitive sequence (IRS) PCR genomics, alleviates many of these constraints. Clonal overlap is detected by hybridization of individual IRS-PCR products to IRS-PCR product pools of the three-dimensional coordinate pools of YAC libraries in dot-blot format. Entire libraries can be screened in a single step, and multiple libraries can be screened simultaneously. Cloning YAC fragments, sequencing, and primer generation are eliminated, increasing the efficiency of contig construction and reducing the expense. In addition, the genomic location of the individual IRS-PCR products can also be simultaneously determined by screening either interspecific backcrosses or radiation hybrid panels, in dot-blot format, confirming contig extension in the region of interest.
Hunter
K
K
Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
D000818:Animals; D002874:Chromosome Mapping / Q000295:instrumentation / Q000379:methods*; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D016678:Genome; D006801:Humans; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid